PERSOONIA Published by Rijksherbarium / Hortus Botanicus, Leiden Volume Part 405-430 15, 4, pp. (1994) Ultrastructure of the ascus apical apparatus in Leotia lubrica and some Geoglossaceae (Leotiales, Ascomycotina) Gerard+J.M. Verkley Rijksherbarium/Hortus Botanicus, P.O. Box 9514, 2300 RA Leiden, The Netherlands The ultrastructure of the apical apparatus and lateral ascus wall is compared in Leotia lubrica and five species currently placed in the Geoglossaceae. The lateral ascus wall consists of two layers, of which the inner one increases in thickness in the apical apparatus. Considerable differences in substructure of both layers are described. On the basis of generalmorphology of the apical apparatus, structure and PA-TCH-SP reactivity of the apical thickening and annulus, and occurrence of an annular protrusion four main categories are distinguished. A reactive annulus is demonstrated in the apical apparatus of all species, including L. lubrica. The species studied are arranged as follows: Category 1a. Geoglossum nigritum and G. cookeianum; 1b. Trichoglossum hirsutum; 2. Leotia lubrica; 3. Microglossum viride; 4. Mitrula paludosa. Most fundamental is considered the position of the annulus in the api- cal thickening, either fully (category 1) or partly (2—4) occupying the apical thickening, either associated with an annular protrusion (3, 4) or not (1, 2). The data on the ultrastruc- ture of the ascus apical apparatus and lateral wall, and mode of dehiscence indicate that L. lubrica takes an isolated position, distant from the other Leotioideae (including Ombro- philoideae) and the Geoglossaceae. Geoglossum, Trichoglossum, and Microglossum can best be maintained as separate genera in the family Geoglossaceae. The ultrastructural data of M.paludosa indicate closer affinity with Sclerotiniaceae than with Geoglossaceae. Introduction The family Geoglossaceae Corda accommodates some of the largest and most con- spicuous forms of the inoperculate discomycetes (Leotiales S. Carp.). It has been the of several studies and of its subject monographical most representatives are relatively well-known (Durand, 1908, 1921; Imai, 1941, 1955, 1956; Nannfeldt, 1942; Mains, 1954, 1955; Eckblad, 1963). Before the fundamental significance of the structure of the ascus apical apparatus became fully appreciated, clavate and capitate inoperculate disco- mycetes were treated as close relatives of operculate discomycetes like Helvella s.l. (Rehm, 1896). The macromorphology of the ascocarp has always been a character of major impor- the of the But other characters tance to taxonomy Geoglossaceae. were investigated more workers like Comer who the and of closely by (1930), compared ontogeny microanatomy stipitate, clavate, and capitate (pileate) ascocarps. Imai (1941) emphasized the shape of spores and the fleshy vs. gelatinous consistency of ascocarps. The Leotioideae, erected by Imaifor Leotia Pers. and NeocudoniellaImai, were transferred to the Helotiaceae by Korf He did the of similarities in and of (1958). so on ground anatomy gelatinization excipulum to certain Ombrophiloideae, particularly Neobulgaria Petr. Later on, Korf (1973) merged 406 PERSOONIA Vol. 15, Part 4, 1994 the Ombrophiloideae into the Leotioideae. Maas Geesteranus (1964) emphasized the taxo- nomic importance of the structure of the stipe and the transitional region of stipe and hyme- nium. Under the influence of these and otherrelevant of the studies, most capitate genera were transferred from the Geoglossaceae to the Leotiaceae Corda (Helotiaceae sensu SclerotiniaceaeWhetz. But the delimitation auct.) or many problems concerning of gen- era, the position of the transferred genera, and the boundary between the Geoglossaceae on the one hand and the Leotiaceae and Sclerotiniaceae on the other remained unsolved (Benkert, 1983). The structure of the ascus, another important source for informative characters, has of hardly been exploited. In particular the ultrastructure the apical apparatus may con- tribute to solving some of these problems. This study aims to determinehow the ultra- structural characters ofthe present Geoglossaceae relate to those ofLeotia lubrica (Scop.) Pers. and other Leotiaceae (Verkley, 1992, 1993b) and Sclerotiniaceae (Verkley, 1993a). Because the study mainly focuses on the relationships within the large family Leotiaceae, the number of taxa had to remain limited. Most of these taxa have been the subject of earlierultrastructural studies (Bellemere, 1975, 1977; Bellemere et al., 1987; Honegger, 1983). But, as explained in previous re- ports, the results are difficult to compare with those on Leotiaceae and Sclerotiniaceae obtainedby Verkley (1992, 1993a, 1993b), and some important data are not yet clarified. For and studied the in Leotia example, Bellemere(1977) Honegger (1983) apical apparatus lubrica, but it remained unclear whether it contains an annulus like the apical apparatus in most other Leotiaceae. Bellemere (1977) and Bellemereet al. (1987) also studiedthe api- cal in apparatus selected Geoglossaceae, Geoglossum spec., Microglossum viride (Pers.) Gillet, Spathularia flavida Pers.: Fr., Mitrulapaludosa Fr., and in Heyderia abietis (Fr.) Link, now residing in the Leotiaceae. New and additional data are presented on ascus wall ultrastructure in Leotia lubrica, Geoglossum nigritum Cooke, G. cookeianum Nannf., Trichoglossum hirsutum (Pers.) Boud., Microglossum viride, and Mitrula paludosa. Trichoglossum hirsutum (Pers.) Boud. is investigated in this way for the first time. The implications ofthe data for the taxonomy of these fungi are discussed. MATERIALS AND METHODS Fresh materialwas collected in the field. Specimens were fixed and embedded in Epon described Ultrathin sections as earlier by Verkley (1992, 1993a). were cut using a dia- mond for PA-TCH-SP knife. Sections were either treated as described earlier (Verkley, 1992), or contrasted with uranyl acetate and lead citrate. Preparations were examined using a Philips EM 300 or Jeol JM 1010electron microscope at 60 kV. In the following list details are given about the origin of the collections, deposited in Leiden (L). Geoglossum nigritum Cooke. Eiland van Rolfers, Amsterdamse Waterleidingduinen, G. prov. Noord-Holland, the Netherlands, in grassland, Oct. 1992, Verkley 153; Ruiter- plaat, Noord-Beveland, prov. Zeeland, the Netherlands, Oct. 1992, G. Verkley 141. Verklcy: Ascus apical apparatus in Leotia lubrica and some Geoglossaceae 407 cookeianum Nannf. Geoglossum Ruiterplaat, Noord-Beveland, prov. Zeeland, the Netherlands, in grassland with moss, Oct. 1992, G. Verkley 140. Trichoglossum hirsutum(Pers.) Boud. Eiland van Rolfers, Amsterdamse Waterleiding- duinen, prov. Noord-Holland, the Netherlands, in grassland, Oct. 1992, G. Verkley 152. Leotia lubrica (Scop.) Pers. Foret de St. Prix, Morvan, dep. de Cote d'Or,France, on the ground in mixed forest, Oct. 1990, J. van Brummelen 7974. viride Gillet. Microglossum (Pers.) Payolle, dep. Hautes Pyrenees, France, on the Oct. J. Brummelen 8020. ground amongst mosses, 1991, van Mitrulapaludosa Fr. Roode Beek, Vlodrop, prov. Limburg, the Netherlands, on plant debris in running water, May 1990, H. Huyser s.n.; Smuddebos, Losser, prov. Over- ijssel, the Netherlands, June 1990,F. Ligtenberg s.n. A detailed clarificationof the terms used for wall structure and stages in ascus develop- ment including the corresponding terms used by Bellemere (1977) and Bellemere et al. (1987) has been given elsewhere (Verkley, 1992). The circumscription of the apical chamberis extended as follows. chamber: of enclosed variable annular Apical amount epiplasm to a extent by an protru- enclosed the of sion, or by most protruding part an apical thickening which is fully occu- pied by an annulus (e.g. Fig. 4). RESULTS For reasons mentionedin earlier work (Verkley, 1992, 1993a, 1993b) the PA-TCH- SP technique was preferred over conventional staining of ultrathin sections for the study of wall substructure. The contrast obtained with this technique is based on the presence in the walls of PAS-positive (periodic acid-Schiff) polysaccharides. Free, vicinal hydroxyl of these oxidized The addition of groups polysaccharides are to aldehyde groups by PA. reduction of TCH to these aldehydes and the subsequent SP by thiocarbohydrazones re- sults in a fine deposit of metallic silver on the thin sections, referred to as 'PAS-reactiv- ity', or, simply, 'reactivity' in this paper. Since most cytoplasmic structures are insuffi- ciently contrasted by this technique, post-staining with uranyl and lead salts was applied for closer study (Figs. 17, 18). Series of longitudinal median sections of young, imma- dehisced asci studied. The lateral the ture, mature, and were ascus wall, apical apparatus, and some special features of the epiplasm are described. GENERAL OBSERVATIONS In the young, elongating ascus initial the apical cytoplasm contains a spherical area (circular in thin section) of microvesicles, surrounded by an area with a variable number of larger apical vesicles. During apex formation, when the apical apparatus is formed (Verkley, 1992), a large concentration of microvesicles containing reactive material is found in the cytoplasm in the direct vicinity of the apical wall (mv, Fig. 3). The species studied all develop two layers in their ascus walls, of which the inner one increases in the But the substructure and differ thickness at apex. reactivity can consider- ably between species. 408 PERSOONIA Vol. 15, Part 4, 1994 409 Verkley: Ascus apical apparatus in Leotia lubrica and some Geoglossaceae